Novel dna aptamers and use thereof

ABSTRACT

The present disclosure relates to novel DNA aptamers and use thereof. In particular, the present disclosure relates to DNA aptamers selected from a DNA library using Cell-SELEX to bind specifically to cancer cells. The DNA aptamers of the present disclosure selected and optimized for high binding affinity to cancer cells can be effectively used for the diagnosis of cancer as they have enhanced targeting efficiencies for target cells and tissues.

TECHNICAL FIELD

The present disclosure relates to novel DNA aptamers. In particular, thepresent disclosure relates to DNA aptamers selected from a pancreaticcancer DNA library using Cell-SELEX to bind specifically to pancreaticcancer cells. The present disclosure is based on a study conducted bothas an original research project of the National Cancer Center (NCC) andpart of the TIPS program (Tech Incubator Program for Startup) of theMinistry of SMEs and Startups.

[Project No.: NCC-1210080, Title: Identification of pancreaticcancer-specific metastasis factors using Cell-SELEX]

[Project No.: NCC-1710400, Title: Study on the regulation of metastasisin pancreatic cancer by the kinase FAM20C secreted into cancermicroenvironment]

[Project No.: 52562351, Title: Development of therapeutic agents forpancreatic cancer using aptamer-antibody-drug conjugates]

BACKGROUND

Aptamers refer to single-stranded DNA or RNA oligonucleotides that haveunique three dimensional structures and specifically bind to targetmolecules in a manner similar to antibodies. In general, aptamers havehigh affinity in concentrations as low as nanomoles to picomoles.

Aptamers are commonly compared to antibodies because of theirtarget-specific binding properties. Compared with antibodies, aptamersare easily produced by chemical synthesis without the need to employbiological processes using cells or animals, relatively stable at hightemperatures, and superior in accessibility to targets owing to theirsmall sizes. In addition, aptamers have advantages over antibodies interms of potential as therapeutic agents in that they can be easilymodified in the course of chemical synthesis and are not immunogenic andnontoxic. However, aptamers have the drawback that they have a shorthalf-life as they are degraded by nucleases in vivo. This drawback canbe overcome by various chemical modifications.

Pancreatic cancer is a carcinoma showing the worst prognosis, with its1-year mortality rate being the highest among all carcinomas. The 2-yearsurvival rate for pancreatic cancer is about 10%, and the 5-yearsurvival rate is only 8% or lower. Over the past two decades, 5-yearsurvival rates in almost all cancers have greatly increased, butpancreatic cancer has given very dismal results, increasing from 3%reported in 1997 to only about 8% in 2016.

Actually, only 20% or so of pancreatic cancer patients are deemed to beoperable, and even in such cases, a high survival rate of 90% or greaterafter operation can be expected only when the tumor size is 1 cm or lesswith no lymph node metastasis or distant metastasis. However, mostpatients are already inoperable at the time of diagnosis. When the tumoris inoperable, patients typically rely on chemotherapies or radiationtherapies. However, since clearly standardized therapies are notcurrently available, early detection of pancreatic cancer is importantin improving the survival rates.

Pancreatic cancer has almost no early symptoms, and by the time apatient can recognize symptoms, the cancer has already progressed to anadvanced stage in most cases. Consequently, early diagnosis ofpancreatic cancer is very difficult. Currently, practically no earlydetection markers are available for pancreatic cancer.

Early pancreatic cancer is generally defined as tumors that have a sizeof less than 2 cm and that are confined to the pancreas with noinfiltrations or lymph node metastases. However, even when the size ofpancreatic cancer is less than 2 cm, the threshold for early pancreaticcancer, metastasis has been found in as many as 50% of such cases. Evenin stage II, which is classified as early pancreatic cancer when stagingpancreatic cancer based on the size of tumor, lymph node metastasis, anddistant metastasis, it is common to find infiltrations in regionsconnecting to the superior mesenteric vein or portal vein, as well asearly metastasis by a small number of cells. In such cases, the tumor isnot resectable. In cases where the tumor has been operated consideringthat distant metastasis was not found on the scan and tumor wasdetermined to be confined to the pancreas, it is not uncommon thatdistant metastasis is found shortly after the operation. In such a case,risk of recurrence is very high even after a surgery, and mediansurvival is only 6˜12 months since there are no anticancer agents withoutstanding efficacy. Given the foregoing, there is a need for detectingearly pancreatic cancer that has not advanced to substantial distantmetastasis or early metastasis by a small number of cells, at least forthe purpose of early surgical resection, the only curative treatment ofpancreatic cancer.

In developing probes specific to cell surface proteins, extracellulardomains of a cell surface protein can be isolated and purified in theform of a recombinant protein and then used as an antigen to developantibodies or for selection of aptamers. The screening process forselection of aptamers is generally called SELEX (systematic evolution ofligands by exponential enrichment) technique. This technique typicallyuses isolated recombinant proteins. However, the three-dimensionalstructure of cell surface proteins is likely to be altered in the courseof isolation, which may lead to a situation where the selected aptamerscannot actually bind to the target protein on the cell surface if thethree-dimensional structure of the protein is critical for the bindingwith the target protein.

Such limitations may be overcome by using the Cell-SELEX method, whichinvolves selecting aptamers cell membrane specifically using livingcells unlike the conventional SELEX technique.

In the present disclosure, DNA aptamers having a high binding affinityto pancreatic cancer were selected using the Cell-SELEX method onpancreatic cancer cells. The selected aptamers have been found to beeffective in cell targeting.

Prior Art Literature

(Patent literature 1) KR 10-1458947

(Patent literature 2) KR 10-1250557

SUMMARY

It is an object of the present disclosure to provide novel DNA aptamers.In particular, the novel DNA aptamers are DNA aptamers that showcancer-specific binding.

It is another object of the present disclosure to provide a method fordiagnosing or treating a cancer using DNA aptamers that showcancer-specific binding.

The present disclosure aims to develop aptamers useful in probing cancercells. Aptamers that specifically bind to cell membranes of pancreaticcancer have been selected using the Cell-SELEX technique. Specifically,in the present disclosure, aptamers that specifically bind to pancreaticcancer cells have been selected using CMLu-1 cells isolated frommetastasized pancreatic cancer tissue as target cells and normalpancreas tissue cell HPNE as control.

In one aspect, the present disclosure provides a DNA aptamer comprisingthe nucleotide sequence of SEQ ID NO: 6. In another aspect, the presentdisclosure provides a DNA aptamer comprising a nucleotide sequencehaving at least 90% or 95% homology to the nucleotide sequence of SEQ IDNO: 6. In one aspect of the present disclosure, the DNA aptamer may showcancer-specific binding. In one aspect of the present disclosure, theDNA aptamer may consist of the nucleotide sequence of SEQ ID NO: 6.

In another aspect, the present disclosure provides a DNA aptamerconsisting of a nucleotide sequence having at least 90% or 95% homologyto the nucleotide sequence of SEQ ID NO: 4. In one aspect of the presentdisclosure, the DNA aptamer may consist of the nucleotide sequence ofSEQ ID NO: 4.

As used herein, “a nucleotide sequence having at least 90% homology”refers to a nucleotide sequence that comprises addition, deletion, orsubstitution of one to several nucleotides to have 90% or greater, butless than 100%, identity in nucleotide sequence relative to a referencesequence and shows similar cancer-specific binding.

In one aspect of the present disclosure, having at least 90% homology tothe nucleotide sequence of SEQ ID NO: 4 may refer to having a nucleotidesequence that differs from the nucleotide sequence of SEQ ID NO: 4 in aregion of SEQ ID NO: 4 other than the region corresponding to thenucleotide sequence of SEQ ID NO: 6.

As used herein, the term “DNA aptamers” refers to short, single-strandedoligonucleotides that bind to corresponding targets with high affinityand specificity and have unique three-dimensional structures. Through aprocess of iterative in vitro selection and enrichment, DNA moleculesthat specifically bind to a specific target, i.e., DNA aptamers, can beselected from a DNA aptamer library.

In an experimental example of the present disclosure, aptamers of a DNApool enriched through the Cell-SELEX process were clustered, on thebasis of their sequence similarity, into 11 aptamer families (SQ1 toSQ11). Among the families, SQ8 has been identified as a group havinghigh binding affinity to CMLu-1, pancreatic cancer-derived metastaticcancer cells.

In addition, using as template the SQ8 (SEQ ID NO: 4) aptamer (80-mer),which specifically binds to pancreatic cancer tissue derived cells, atruncated aptamer (28-mer) was prepared in order to enhance synthesisyields and reduce costs of synthesis. Specifically, the SQ8-4 aptamer(SEQ ID NO: 6) was prepared, which reduces the size of aptamer bygreater than one half while retaining the binding affinity to CMLu-1target cells. The inventors of the present disclosure concluded that theSQ8-4 aptamer is a region of the SQ8 sequence which is critical for itsability to target cancer cells and cancer tissue, and based thereon,conducted further experiments.

The target cells used for Cell-SELEX in the working examples of thepresent disclosure were CMLu-1 cells, which were obtained frommetastatic pancreatic cancer tissue through an orthotopic graftexperiment. Accordingly, the present disclosure may be usefulparticularly for the diagnosis of pancreatic cancer.

The present disclosure provides a composition for targeting cancertissues, comprising a DNA aptamer described above. In one aspect of thepresent disclosure, the composition comprising a DNA aptamer may furthercomprise, in addition to the above component, an active ingredienthaving the same or similar function, or a component that stabilizes theformulation of the composition or enhances the stability of the aptamer.In one aspect of the present disclosure, the composition may be apharmaceutical composition.

In addition, the present disclosure provides a composition fordiagnosing a cancer comprising an aptamer described above.

In one aspect of the present disclosure, a DNA aptamer can be used incombination with an effector moiety.

In one aspect of the present disclosure, the effector moiety may be acytotoxic agent, an immunosuppressive agent, an imaging agent (e.g., afluorescent moiety or chelator), a nanomaterial or a toxin polypeptide.The cytotoxic agent may be a chemotherapeutic agent.

In one aspect, the present disclosure relates to composition fortreating a cancer, comprising a novel DNA aptamer according to an aspectof the present disclosure and an anticancer agent conjugated with theDNA aptamer.

In one aspect of the present disclosure, the cancer may be pancreaticcancer, colon cancer, liver cancer, lung cancer, brain tumor, oralcavity cancer, or breast cancer, but is not limited thereto.

In one aspect of the present disclosure, the anticancer agent may be oneor more selected from the group consisting of MMAE(monomethyl auristatinE), MMAF(monomethyl auristatin F), calicheamicin, mertansine (DM1),ravtansine (DM4), tesirine (SCX), doxorubicin, cisplatin, SN-38,duocarmycin, and (yrrolobenzodiazepine (PBD), but is not limitedthereto.

In one aspect of the present disclosure, the DNA aptamer may beconjugated with a polyethylene glycol (PEG) or its derivative, adiacylglycerol (DAG) or its derivative, a dendrimer, or a zwitterion-containing biocompatible polymer (e.g., aphosphorylcholine-containing polymer).

In one aspect of the present disclosure, the composition may furthercontain a physiologically acceptable excipient, carrier, or additive,which may include, but is not limited to, starch, gelatinized starch,microcrystalline cellulose, lactose, povidone, colloidal silicondioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gumarabic, pregelatinized starch, corn starch, cellulose powder,hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax,synthetic aluminum silicate, stearic acid, magnesium stearate, aluminumstearate, calcium stearate, white sugar, dextrose, sorbitol, and talc.

In one aspect of the present disclosure, the composition may beadministered to subjects in a variety of forms according to the selectedroute of administration as understood by a person of ordinary skill inthe relevant technical fields. For example, a composition of the presentdisclosure may be administered by topical, enteral, or parenteralapplication. Topical application includes, but is not limited to,application to epidermis, inhalation, enema, eye drop, ear drop, andmucosal application. Enteral application includes oral administration,rectal administration, vaginal administration, and gastric feeding tube.Parenteral administration includes, but is not limited to, intravenous,intra-arterial, intracapsular, intraorbital, intracardiac,intracutaneous, transtracheal, subcuticular, intra-articular,subcapsular, subarachnoid, intramedullary, epidural, intrastemal,intraperitoneal, subcutaneous, intramuscular, transepithelial,intranasal, intrapulmonary, intrathecal, rectal, and topicaladministration.

In addition, in one aspect of the present disclosure, the compositionmay be formulated into any forms suitable for the selected route ofadministration. For formulation purposes, the composition may beprepared using diluents or excipients including, but not limited to, afiller, an extender, a binder, a wetting agent, a disintegrant, asurfactant, and the like.

In one aspect of the present disclosure, the composition may contain aDNA aptamer according to an aspect of the present disclosure in anamount determined by a person of ordinary skill in the art to beeffective considering the route of administration as well as the weight,age, sex, health condition, diet, time of intake, and excretion rateetc. of the subject in need of administration.

The DNA aptamers of the present disclosure selected and optimized forhigh binding affinity to cancer cells can be effectively used for thediagnosis and treatment of cancer as they have enhanced targetingefficiencies for target cells and tissues.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the format of the nucleotide sequences included inthe DNA library of the present disclosure as well as the formats of theforward primer and reverse primer that can be used to amplify oridentify the above nucleotide sequences. Specifically, the nucleotidesequences in the DNA library comprise 20 constant nucleotides at the5′-terminus, 40 random nucleotides in the middle, and additional 20constant nucleotides at the 3′-terminus. The forward primer is labeledwith Cy5 at its 5′-terminus (5′-Cy5-sequence-3′), and the reverse primerwith biotin at its 5′-terminus (5′-biotin-sequence-3′).

FIG. 2 shows the results of enriching a ssDNA pool, out of the DNAlibrary of the present disclosure, which has binding affinity topancreatic cancer cells and determining with a flow cytometer(Fluorescence-activated cell sorting; FACS) whether the cell bindingaffinity of the pool is increased according to the number of rounds.

FIG. 3 schematically illustrates the process of screening aptamersthrough Cell-SELEX technique using metastatic pancreatic cancer cells inthe present disclosure.

FIG. 4 shows the results from the determination of cell binding affinityof individual Cy5-labeled aptamer candidates obtained by screeningaptamers through Cell-SELEX technique using metastatic pancreatic cancercells in the present disclosure.

FIG. 5 depicts the secondary structure determined for the SQ8 aptamer(Example 1) obtained by screening aptamers through Cell-SELEX techniqueusing metastatic pancreatic cancer cells in the present disclosure.

FIG. 6a shows the results from the determination of target cell bindingaffinity of the SQ8 aptamer of the present disclosure using a flowcytometer (FACS). Specifically, target cell binding was determined forExample 1 (SQ8 aptamer) as well as no-treatment control and ComparativeExample 1 (DNA pool library).

FIG. 6b shows the results from the determination of target cell bindingaffinity of the SQ8-4 aptamer of the present disclosure using a flowcytometer (FACS). Specifically, target cell binding was determined forExample 1 (SQ8 aptamer) and Example 2 (SQ8-4 aptamer) as well asno-treatment control and Comparative Example 1 (DNA pool library).

FIG. 7 depicts the secondary structure of the SQ8-4 aptamer (Example 2),which was prepared based on the SQ8 aptamer (Example 1) of the presentdisclosure.

FIG. 8 shows targeting profile of Example 1 (SQ8 aptamer) of the presentdisclosure for target cells determined with confocal microscopy. Grayellipses represent nuclei, and the brightest looking, white areasrepresent aptamers that are bound to cell surfaces or internalized intocells.

FIG. 9 shows targeting profile of Example 2 (SQ8-4 aptamer) of thepresent disclosure for target cells determined with confocal microscopy.Gray ellipses represent nuclei, and the brightest looking, white areasrepresent aptamers that are bound to cell surfaces or internalized intocells.

FIG. 10 shows targeting profile of Example 1 (SQ8 aptamer) of thepresent disclosure for pancreatic cancer tissue determined withbioluminescence imaging in a xenograft mouse model for a humanpancreatic cancer cell line.

FIG. 11 shows targeting profile of Example 2 (SQ8-4 aptamer) of thepresent disclosure for pancreatic cancer tissue determined withbioluminescence imaging in a xenograft mouse model for a humanpancreatic cancer cell line.

FIG. 12 shows targeting profile of Example 2 (SQ8-4 aptamer) of thepresent disclosure for pancreatic cancer tissue determined withbioluminescence imaging in a xenograft mouse model for pancreatic cancercells of a human pancreatic cancer patient.

FIG. 13 shows binding affinities of the SQ8-4 aptamer of the presentdisclosure to various pancreatic cancer cell lines determined with aflow cytometer (FACS). Specifically, cell binding was determined forExample 2 (SQ8-4 aptamer) as well as no-treatment control andComparative Example 3 (SQ8-4-Comp aptamer).

FIG. 14 shows the geometric means of the relative fluorescenceintensities of Example 2 over Comparative Example 3 in number of folds,based on the results given in FIG. 13.

FIG. 15a and FIG. 15b show binding affinities of the SQ8-4 aptamer ofthe present disclosure to various cancer cell lines determined with aflow cytometer (FACS). Specifically, cell binding was determined forExample 2 (SQ8-4 aptamer) as well as no-treatment control andComparative Example 3 (SQ8-4-Comp aptamer).

FIG. 16 shows the geometric means of the relative fluorescenceintensities of Example 2 over Comparative Example 3 in number of folds,based on the results given in FIG. 15a and FIG. 15 b.

FIG. 17 shows binding affinities of the SQ8-4 aptamer of the presentdisclosure to various PDOX-derived cell lines determined with a flowcytometer (FACS). Specifically, cell binding was determined for Example2 (SQ8-4 aptamer) as well as no-treatment control and ComparativeExample 3 (SQ8-4-Comp aptamer).

FIG. 18 shows the geometric means of the relative fluorescenceintensities of Example 2 over Comparative Example 3 in number of folds,based on the results given in FIG. 17.

DETAILED DESCRIPTION

The present disclosure will be clearly understood from the aspectsdescribed above and Experimental Examples or Examples described below.In the following, the present disclosure will be explained in detailsuch that a person of ordinary skill in the art can easily understandand reproduce the disclosure, by way of working examples described inreference to accompanying tables. However, the Experimental Examples orExamples described below are given just to illustrate the presentdisclosure and the scope of the present disclosure is not limited tosuch Experimental Examples or Examples.

Experimental Example 1 Aptamer Screening Using Cell-SELEX

Experimental procedures for screening of aptamers that specifically bindto pancreatic cancer cells using the Cell-SELEX technique areschematically illustrated in FIG. 3.

Specifically, to obtain a cell line expressing cell membrane proteinscharacteristic of pancreatic cancer, pancreatic cancer cells weretransplanted into an animal model and pancreatic cancer cell line CMLu-1was isolated from a tissue to which the cancer had metastasized (FIG.3A).

A ssDNA library was prepared and then screened using cells of thepancreatic cancer cell line CMLu-1 as the target cells (positive cells)and hTERT/HPNE cells (Human Pancreatic Nestin Expressing cells) as thecontrol cells (negative cells). Selection of ssDNA molecules which bindonly to the pancreatic cancer cells and not to the control cells wasiterated for multiple rounds, and the resulting enriched ssDNA pool wascloned and sequenced, followed by clustering.

(1) Construction of a metastatic pancreatic cancer cell line from axenograft mouse model of a human pancreatic cancer cell line

The metastatic pancreatic cancer cell line CMLu-1 was obtained asdescribed below. An orthotopic mouse model was built using NOD/SCIDmouse. First, to construct an animal model that can mimic metastasis ofpancreatic cancer, a pancreatic cancer cell line stably expressing thefirefly luciferase (CFPAC-1-Luci) was established and used to enablenon-invasive monitoring of tumorigenesis over time. CFPAC-1-Lucipancreatic cancer cells were transplanted orthotopically into a NOD/SCIDmouse. After 43 days, tumor tissue was removed from the lung tissue ofthe mouse where pancreatic cancer had metastasized, and the isolatedtumor tissue was genotyped, showing that the genetic attributes of themetastatic tumor cells are identical to those of the pancreatic cancercells. Then, single cells were prepared from the tumor tissue andcultured. CMLu-1 cells isolated from the metastatic tumor tissue werecultured and maintained in RPMI-1640 medium (Hyclone, Logan, Utah, USA)plus 10% FBS (Thermo Fisher Scientific, USA) and 100 IU/mL of Anti-Anti(antibiotic-antimycotic; Gibco).

The resulting CMLu-1 cells were used as the cells for positive selectionin Cell-SELEX, and human pancreatic duct normal epithelial cells (HPNE)purchased from ATCC Inc. were used as the control cells for negativeselection.

(2) Preparation of a ssDNA library and primers for Cell-SELEX

The DNA library used in Cell-SELEX for pancreatic cancer-specificaptamers was a pool of DNA sequences that are composed of a combinationof constant and unique nucleotides. The DNA sequences comprised 20constant nucleotides at the 5′-terminus, 40 random nucleotides in themiddle, and additional 20 constant nucleotides at the 3′-terminus.5′-terminus of the DNA sequences was labeled with Cy5 in order tomonitor enrichment of selection using a fluorescence-activated cellsorter (“FACS”; so-called “flow cytometer”), and the 3′-terminus waslabeled with biotin for purification of the ssDNA molecules (FIG. 1). Inaddition, the forward primer was labeled with Cy5 at its 5′-terminus(5′-Cy5-sequence-3′), and the reverse primer with biotin at its5′-terminus (5′-biotin-sequence-3′). The compositions of DNAs includedin the DNA library as well as the forward and reverse primers are asshown in Table 1 below.

TABLE 1 Format of DNA 5′-ATA CCA GCT TAT TCA ATT- library[nucleotides 40(N40)]-  nucleotides AGA TAG TAA GTG CAA TCT-3′(SEQ ID NO: 1) Forward primer; 5′-Cy5-ATA CCA GCT TAT TCA ATT-3′5′-primer (SEQ ID NO: 2) Reverse primer; 5′-biotin-AGA TTG CAC TTA CTA 3′-primer  TCT-3′ (SEQ ID NO: 3)

PCR was used to amplify eluted DNA pools. ssDNAs were isolated bycapturing biotinylated complementary strands using thestreptavidin-biotin bond and denaturing double-stranded DNAs with NaOH.PCR mixes were prepared, and PCR was carried out as instructed by themanufacturer.

(3) Library screening through Cell-SELEX

The ssDNA library prepared as described above was screened using CMLu-1cells as the target cells (positive cells) and hTERT/HPNE cells as thecontrol cells (negative cells).

10 nmol of the DNA library was dissolved in 1,000 μL of a binding buffer(Dulbecco's PBS (Hyclone, USA) with 5 mM MgCl₂, 0.1 mg/mL tRNA, and 1mg/mL BSA). The DNA library or enriched pool was denatured at 95° C. for10 min and cooled on ice for 10 min, followed by incubation with CMLu-1cells in an orbital shaker at 4° C. for 1 hour. The CMLu-1 cells werethen washed 3 times to remove unbound DNA sequences, and the bound DNAmolecules were eluted via centrifuge using 1,000 μL of a binding bufferat 95° C. for 15 min. To carry out a counter selection, an aptamer poolwas incubated with hTERT/HPNE cells for 1 hour, after which thesupernatant was collected for negative selection. The enriched poolswere monitored using FACS, and Quiagen's cloning kit for sequencing(Quiagen, Germany) was used for cloning into Escherichia coli toidentify aptamer candidates.

(4) Cloning and sequencing of enriched ssDNA pool and multiple sequencealignment

For selection of candidate sequences, the enriched ssDNA pool after 5rounds was cloned and sequenced. The ssDNA pool was amplified by PCRusing unmodified primers, ligated to pGEM-T easy vector (Promega, USA),and then cloned into HIT™-DH5a competent cells (Promega, USA).Thereafter, 200 cloned sequences were analyzed by Cosmogenetech Inc.(Seoul, Korea) and aligned using ClustalX 1.83.

The extent of enrichment according to the number of rounds of selectionis as illustrated in FIG. 2.

The process described above in (1) to (4) is schematically illustratedin FIG. 3. Aptamers sequenced through the above process were clusteredinto aptamers with similar sequences. As a result, eleven Cy5-labeledaptamer family candidates (SQ1 to SQ11) were identified. The contents ofindividual aptamer families in the entire pool were as shown in Table 2below.

TABLE 2 Content in Aptamer enriched DNA family pool (%) SQ1  14.09 SQ2 13.42 SQ3   5.37 SQ4   4.70 SQ5   4.70 SQ6   3.36 SQ7   2.01 SQ8   2.68SQ9   1.34 SQ10  1.34 SQ11  1.34

Experimental Example 2 Determination of Target Cell Binding Specificityof Enriched Aptamer Family Candidates

The CMLu-1 target cell binding specificities of the enriched aptamerfamily candidates obtained in (4) of Experimental Example 1 weredetermined with flow cytometry (FACS).

Each Cy5-labeled aptamer family candidate was incubated, along with3×10⁵ CMLu-1 cells and hTERT/HPNE cells, in the binding buffer used forCell-SELEX at 4° C. for 1 hour. The cells were washed 3 times withbinding buffer containing 0.1% NaN₃, and the pellets having boundsequences were resuspended in the binding buffer. Fluorescence-basedassay was carried out on 10,000 cells using BD FACSCallibur™ andFACSVerse™ (BD Biosciences, USA), and the data were analyzed usingFlowJo software v10.0.7.

The results from the determination of target cell binding affinity ofindividual Cy5-labeled aptamer family candidates are shown in FIG. 4. Anaptamer with a high binding specificity for metastatic pancreatic cancercells (CMLu-1), the target cells, was identified as SQ8, whose sequenceis shown below.

*SQ8 aptamer sequence (SEQ ID NO: 4)5′-AGCAGCACAGAGGTCAGATGCTTGGGCTATTTCTTATTCATGCTGTTCCACCGCTCTCGGCCTATGCGTGCTACCGTGAA-3′

Experimental Example 3 Analysis of SQ8 Aptamer and FunctionalCharacterization of Aptamer Fragments

The secondary structure determined for the SQ8 aptamer selected inExperimental Example 2 is as shown in FIG. 5. In addition, to confirmthe cell binding affinity, target cell binding affinity was determinedfor the SQ8 aptamer (Example 1), no-treatment control, and ComparativeExample 1 (DNA pool library) in the same manner as in ExperimentalExample 2 using FACS. As demonstrated in FIG. 6a , the results show thatwhereas the control and Comparative Example 1 had low binding affinitiesof similar levels, the SQ8 aptamer of Example 1 exhibited a remarkablysuperior target cell binding affinity.

In order to determine whether some portions of the SQ8 aptamer arecritical for the pancreatic cancer-specific binding, various aptamerscomprising parts of SQ8 were prepared and their binding affinity to thetarget cell was investigated. If the length of an aptamer can be reducedwhile retaining its cell binding affinity, it is likely that aptamerproduction costs are reduced while cell penetration is enhanced. Theresults demonstrated that the SQ8-4 aptamer (Example 2) having thesequence indicated in Table 3 below is superior in terms of endocytosiswhile almost fully retaining the target cell binding affinity.Specifically, target cell binding affinities of Example 1, Example 2,no-treatment control, and Comparative Example 1 (DNA pool library) weredetermined in the same manner as in Experimental Example 2 using FACS.As demonstrated in FIG. 6b , the results show that whereas the controland Comparative Example 1 had low binding affinities of similar levels,the SQ8 aptamer of Example 1 exhibited a remarkably superior target cellbinding affinity, and the SQ8-4 aptamer of Example 2 was superior evento Example 1.

The nucleotide sequence of SQ8-4 is shown below (SEQ ID NO: 6), and thecorresponding secondary structure is shown in FIG. 7. In addition,Comparative Example 2 (SQ8-Comp; an aptamer having a nucleotide sequencepartly complementary to SQ8; SEQ ID NO: 5) and Comparative Example 3(SQ8-4-Comp; an aptamer having a nucleotide sequence complementary toSQ8-4; SEQ ID NO: 7) were prepared in order to use them as controls forExamples 1 and 2 in subsequent experiments.

TABLE 3 Aptamer Sequence Example 1 5′-AGCAGCACAGAGGTCAGATGCTTGGGCTATTTCT(SQ8) TATTCATGCTGTTCCACCGCTCTCGGCCTATGCGTGC TACCGTGAA-3′ (SEQ ID NO: 4)Comparative 5′-AGCAGCACAGAGGTCAGATGGAACCCGATAAAGA Example 2ATAAGTACGACAAGGTGGCGAGAGCCCCTATGCGTGC (SQ8-Comp)TACCGTGAA-3′ (SEQ ID NO: 5) Example 2 5′-AGCAGCACAGAGGTCAGATGCTTGGGCT-3′(SQ8-4) (SEQ ID NO: 6) Comparative  5′-TCGTCGTGTCTCCAGTCTACGAACCCGA-3′Example 3 (SEQ ID NO: 7) (SQ8-4- Comp)

Experimental Example 4 Determination of Efficient Targeting of theSelected Aptamer into Cells and Tissues

(1) Endocytosis efficiencies of selected aptamers were determined byconfocal microscopy imaging.

1×10⁴ cells/well of control cells (HPNE) and target cells (CMLu-1) wereplated on 8-well chamber slides (Thermo scientific, USA) coated withpoly-L-lysine (Sigma, USA) 4 hours prior to the experiment. Upon washingwith a washing buffer, 250 nM of a Cy5-labeled aptamer (i.e., Example 1,Example 2, Comparative Example 2, and Comparative Example 3) orComparative Example 1 (DNA pool library) was added to 200 μl of bindingbuffer at 4° C. and incubated. After washing twice, the cells were fixedusing 4% paraformaldehyde, followed by staining of the nucleus withHoechst33342. Thereafter, the cells were subjected to imaging byconfocal microscopy (LSM780, Carl Zeiss, Germany), and the images thusobtained were analyzed with the Zen blue software.

Results of confocal microscopy on Examples 1 and 2 are as shown in FIGS.8 and 9. The brightest looking, white areas in the pictures representregions heavily populated with aptamers. As seen in FIGS. 8 and 9, theaptamers of Examples 1 and 2 targeted pancreatic cancer cells over thecontrol cells and showed excellent levels of endocytosis(internalization) into the cells. The aptamers of Examples 1 and 2 werealso remarkably superior to those of the Comparative Examples intargeting pancreatic cancer cells and showed superior levels ofendocytosis into the cells.

(2) Determination of pancreatic cancer targeting by aptamers through invivo and ex vivo fluorescence imaging

Female Hsd: Athymic nude-Foxn1 nude mice aged 6 weeks were purchasedfrom Harlan Laboratories, Inc. (France). The mice were housed in aspecific pathogen free (SPF) environment under controlled conditions oflight and humidity, with their food and water supplied by the NCC animalfacility. All animal studies were reviewed and approved by theInstitutional Animal Care and Use Committee (IACUC) of the NationalCancer Center Research Institute (NCCRI) (NCC-16-247). The NCCRI is afacility accredited by the Association for Assessment and Accreditationof Laboratory Animal Care International (AAALAC International).

An orthotopic xenograft mouse model of pancreatic cancer was constructedby injecting CFPAC-1 cells (1×10⁶ cells) purchased from the ATCC intothe tail of mouse pancreas. At 3 weeks after inoculation, mice weredivided into two groups according to the treatment to be given, that is,Cy5.5-labeled Example 1 and Comparative Example 2 or Cy5.5-labeledExample 2 and Comparative Example 3, followed by intravenousadministration of a Cy5.5-labeled aptamer (300 pmol/50 μl PBS) mentionedabove.

For ex vivo experiments, mice were sacrificed 15 min or 3 hours afteradministration and then dissected. Tumor tissues were removed andsubjected to bioluminescence imaging using IVIS Lumina (Caliper LifeScience, Hopkinton, Mass., USA). All image data were analyzed usingLiving Image Acquisition and Analysis software.

The gray-scale pictures in FIGS. 10 and 11 show the results ofbioluminescence imaging for Examples 1 and 2. Total flux obtained fromthe imaging results are plotted in FIGS. 10 and 11.

In the imaging results presented at the top of the above drawings, thebrighter (whiter) a region appears, the greater the amount of aptamersbound to the cells in that region is. It can be seen that the imagingresults for the aptamers of Examples 1 and 2 appear whiter than thosefor Comparative Examples 2 and 3. In contrast, the imaging results forComparative Examples 2 and 3 do not show any white regions.

Thus, it has been demonstrated that the aptamers of the presentdisclosure move towards and bind specifically to pancreatic cancertissue compared with aptamers of the Comparative Examples and haveremarkably superior targeting efficiencies for pancreatic cancertissues.

Experimental Example 5 Determination of Targeting in a Mouse OrthotopicXenograft Model for Human Patient Pancreatic Cancer Tissue(Patient-Derived Orthotopic Xenograft Model; PDOX Model)

In order to determine whether the aptamers of the present disclosureexhibit excellent targeting efficiencies even in a PDOX model which canrecapitulate the complexity and heterogeneity of patient tumor tissue,in vivo verification experiments were carried out using fluorescenceimaging.

With the approval of the Institutional Review Board (“IRB”) of the NCC,a patient-derived orthotopic xenograft (PDOX) model was established bycollecting specimens from patients who submitted informed consent anddirectly transplanting them into the pancreas of a nude mouse (purchasedfrom Harlan Laboratories, Inc. (France)). As for primary tumor specimensfrom pancreatic cancer patients (called “HPTs” below), right uponsurgical resection, and in the case of patients with inoperable advancedpancreatic cancer, right after collection of specimens for livermetastasis tissue biopsy (called “GUNS” below) from the patients,specimens were transported in tubes containing a medium and transplantedas soon as possible into female Hsd: Athymic nude-Foxn1 nude mice(obtained from the same source as in Experimental Example 4), by makingan incision in the tail of pancreas and then closing the incision aftertransplantation (PDOX 1St generation, F1). Thereafter, the size of tumorwas measured periodically using abdominal palpation and MRI, and whenthe tumor attains a volume of 3000 mm³, the mouse was sacrificed toobtain tumor tissue. Tumor tissue fragments of a certain size (3 mm*3mm*3 mm) were then orthotopically re-implanted into multiple nude micesubjects to generate subsequent generations (F2, F3, F4 . . . ) andincrease the number of subjects.

At the 4^(th) generation (F4), the established PDOX mouse model wasdivided into two groups of three subjects, and the individual groupswere given intravenously either Example 2 or Comparative Example 3, inthe form of Cy5.5-labeled aptamer (300 pmol/50 μl PBS). 15 min after theadministration, the mice were sacrificed and dissected to remove tumortissues, which were then subjected to bioluminescence imaging using IVISLumina (Caliper Life Science, Hopkinton, Mass., USA). All image datawere analyzed using Living Image Acquisition and Analysis software.

The gray-scale picture in FIG. 12 shows the results of bioluminescenceimaging for Example 2. Total flux obtained from the imaging results areplotted in FIG. 12.

As in FIGS. 10 and 11, in the imaging results presented at the top ofFIG. 12 also, the brighter (whiter) a region appears, the greater theamount of aptamers bound to the cells in that region is. It can be seenthat the aptamer of Example 2 gives a much whiter image than the aptamerof Comparative Example 3. In contrast, the imaging result forComparative Example 3 does not show any white regions.

Thus, it has been demonstrated that the aptamers of the presentdisclosure have remarkably superior targeting efficiencies even in aPDOX model which retains the complexity and heterogeneity of patienttumor tissue.

Experimental Example 6 Determination of Binding Affinity in VariousPancreatic Cancer Cell Lines

Additional flow cytometry (FACS) assays were carried out to determinethe binding affinity of the aptamers of the present disclosure tovarious pancreatic cancer cell lines.

Flow cytometry was carried out in the same manner as in ExperimentalExample 2, using Cy5-labeled aptamers of Example 2 and ComparativeExample 3 at a concentration of 250 nM. Cells from CFPAC-1 cell line,Capan-1 cell line, HPAF-II cell line, MIA PaCa cell line, BxPC-3 cellline, and PANC-1 cell line were used. All of the above-mentioned celllines were purchased from the ATCC.

Results from the determination of binding affinity of the aptamers tovarious pancreatic cancer cell lines are shown in FIGS. 13 and 14. FIG.14 shows the geometric means of the relative fluorescence intensities ofExample 2 over Comparative Example 3 in number of folds.

According to the results shown in FIGS. 13 and 14, the aptamer ofExample 2 of the present disclosure binds more efficiently andspecifically to all the pancreatic cancer cell lines compared with theaptamer of Comparative Example 3. Taken together with the results ofExperimental Examples 4 and 5 discussed above, it can be seen that theaptamers of the present disclosure would specifically bind to varioustypes of pancreatic cancer cells.

In addition, the aptamer of Example 1 similarly would specifically bindto various types of pancreatic cancer cells as it comprises the aptamersequence of Example 2.

Experimental Example 7 Determination of Binding Affinity in VariousTumor Cell Lines

Additional flow cytometry (FACS) assays were carried out to determinethe binding affinity of the aptamers of the present disclosure tovarious tumor cell lines.

Flow cytometry was carried out in the same manner as in ExperimentalExample 2, using Cy5-labeled aptamers of Example 2 and ComparativeExample 3 at a concentration of 250 nM. Cells from HTC116 cell line,Hep3B cell line, A549 cell line, U87 cell line, CAL27 cell line,MDA-MB231 cell line, MCF7 cell line, and KPL4 cell line were used. Allof the above-mentioned cell lines were purchased from the ATCC.

Results from the determination of binding affinity of the aptamers tovarious cancer cell lines are shown in FIGS. 15a, 15b , and 16. FIG. 16shows the geometric means of the relative fluorescence intensities ofExample 2 over Comparative Example 3 in number of folds.

According to the results shown in FIGS. 15a, 15b , and 16, the aptamerof Example 2 of the present disclosure binds more efficiently andspecifically to various cancer cell lines such as colon cancer, livercancer, lung cancer, brain tumor, oral cavity cancer, and breast cancercell lines, compared with the aptamer of Comparative Example 3. Takentogether with the results of Experimental Examples 4 and 5 discussedabove, it can be seen that the aptamers of the present disclosure wouldspecifically bind to various types of cancer cells.

In addition, the aptamer of Example 1 similarly would specifically bindto various types of cancer cells as it comprises the aptamer sequence ofExample 2.

Experimental Example 8 Determination of Binding Affinity in PancreaticDuctal Adenocarcinoma PDOX-derived Cell Lines

Additional flow cytometry (FACS) assays were carried out to determinewhether the aptamers of the present disclosure are likely to bindspecifically to pancreatic cancer cells of clinical patients.

Unlike normal cells, which only have a limited number of cell divisionsbefore death, cancer cells have the characteristic of infinitedivisions. Accordingly, cancer cells isolated from tumor tissue arebelieved to be able to form a cell line capable of proliferatinginfinitely even in the absence of transfection and recapitulate clinicaland molecular biological characteristics of the patient. In thisexperiment, tumor tissue removed from a pancreatic cancer PDOX mouse wasdivided into 3 mm*4 mm fragments, mixed with a human cell dissociationkit (Miltenyi Biotech Inc.) comprising collagenase, and reacted in atissue dissociator (Gentle Macs, Miltenyi Biotech Inc) for 1 hour toseparate the cells from connective tissue. Upon completion of thereaction, RPMI medium containing fetal bovine serum (FBS) was added toinhibit enzymatic activities, followed by centrifugation, to giveprecipitates of cells dissociated from tissue. The precipitates weresuspended in RPMI medium containing FBS, and the cells were then platedevenly on 10 cm petri dishes to a level of 2×10⁶ cells. The medium wasreplaced every other day while removing normal fibroblasts and deadcell, thereby establishing a PDOX-derived cancer cell line for eachpancreatic cancer patient. The established cell lines were named in thesame manner as the PDOX from which they were derived.

On cancer cell lines isolated from tumor tissues of PDOX models usingliver metastasis patient biopsy tissues (GUN#38 and GUN#41) and usingsurgical resection specimens (HPT#19, HPT#22, and HPT#43), flowcytometry was carried out in the same manner as in Experimental Example2, using Cy5-labeled aptamers of Example 2 and Comparative Example 3 ata concentration of 250 nM.

Results from the determination of binding affinity of the aptamers tovarious PDOX-derived cell lines are shown in FIGS. 17 and 18. FIG. 18shows the geometric means of the relative fluorescence intensities ofExample 2 over Comparative Example 3 in number of folds.

According to the results shown in FIGS. 17 and 18, the aptamer ofExample 2 of the present disclosure binds more efficiently andspecifically to various PDOX-derived cell lines which have been derivedfrom pancreatic cancer tissues collected from different patients,compared with the aptamer of Comparative Example 3. Taken together withthe results of Experimental Examples 4 and 5 discussed above, it can beseen that the aptamers of the present disclosure would specifically bindto pancreatic cancer tissue of clinical patients.

In addition, the aptamer of Example 1 similarly would specifically bindto pancreatic cancer tissue of patients as it comprises the aptamersequence of Example 2.

Although the technical idea of the present disclosure has been describedabove by referring to embodiments described in working examples andillustrated in the accompanying drawings, it should be noted thatvarious substitutions, modifications, and changes can be made withoutdeparting from the technical idea and scope of the present disclosurewhich can be understood by a person of ordinary skill in the art towhich the present disclosure pertains. In addition, it should be notedthat that such substitutions, modifications and changes are intended tobe encompassed by the scope of the appended claims.

1. A DNA aptamer comprising a nucleotide sequence having at least 90%homology to the nucleotide sequence of SEQ ID NO:
 6. 2. The DNA aptamerof claim 1, wherein the aptamer comprises the nucleotide sequence of SEQID NO:
 6. 3. The DNA aptamer of claim 1, wherein the aptamer consists ofthe nucleotide sequence of SEQ ID NO:
 6. 4. The DNA aptamer of claim 1,wherein the aptamer consists of a nucleotide sequence having at least90% homology to the nucleotide sequence of SEQ ID NO:
 4. 5. The DNAaptamer of claim 1, wherein the aptamer consists of the nucleotidesequence of SEQ ID NO:
 4. 6. A composition for targeting cancer tissues,comprising the DNA aptamer of claim
 1. 7. A composition for diagnosing acancer, comprising the DNA aptamer of claim
 1. 8. A composition fortreating a cancer, comprising the DNA aptamer of claim
 1. 9. Thecomposition of claim 8, wherein the cancer is pancreatic cancer, coloncancer, liver cancer, lung cancer, brain tumor, oral cavity cancer, orbreast cancer.
 10. The composition of claim 8, further comprising ananticancer agent conjugated with the DNA aptamer.
 11. The composition ofclaim 10, wherein the anticancer agent is one or more selected from thegroup consisting of MMAE (monomethyl auristatin E), MMAF (monomethylauristatin F), calicheamicin, mertansine, ravtansine, tesirine,doxorubicin, cisplatin, SN-38, duocarmycin, and pyrrolobenzodiazepine.12. The composition of claim 8, wherein the DNA aptamer is conjugatedwith a polyethylene glycol (PEG) or its derivative, a diacylglycerol(DAG) or its derivative, a dendrimer, or a phosphorylcholine-containingpolymer.
 13. A method of targeting cancer tissues, comprisingadministering the DNA aptamer of claim 1 to a subject in need thereof14. A method of diagnosing a cancer, comprising administering the DNAaptamer of claim 1 which is conjugated with an imaging agent to asubject in need thereof, and detecting a signal released by the imagingagent to diagnose the cancer.
 15. A method of treating a cancer,comprising administering the DNA aptamer of claim 1 which is conjugatedwith an anticancer agent to a subject in need thereof.